Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is etiologically linked with KS, primary effusion lymphomas (PELs) and multicentric Castleman's disease. These diseases occur in AIDS and other patients and current therapies are limited. KSHV latently infects the vast majority of tumor cells and viral DMA persists as a multiple copy, extrachromosomal, circular episome. To persist in proliferating cells, episomes must replicate and efficiently segregate to daughter nuclei. KSHV latency associated nuclear antigen (LANA) binds terminal repeat (TR) DNA to mediate KSHV episome persistence. LANA mediates TR DMA replication and tethers episomes to mitotic chromosomes for efficient segregation to progeny nuclei. Significant gaps remain in our understanding of LANA mediated episome maintenance. LANA chromosome association is essential for episome tethering yet the mechanisms underlying chromosome attachment are ill-defined. LANA is a large, multifunctional protein. Although LANA shares sequence homology and episome maintenance function with LANA homologs of other gamma-2 herpesviruses, it is much larger than the other LANA homologs. Definition of the LANA domains required for episome maintenance is central to understanding KSHV episome persistence. The precise cis-acting TR sequence requirements for LANA mediated DNA replication and episome maintanence are unknown. Since TRs account for -20% of the KSHV genome, an understanding of the necessary cis-acting episome persistence sequence is fundamental to a better understanding of KSHV biology. This work will investigate critical aspects of LANA's episome maintenance function. The mechanisms by which the LANA N- and C-termini independently associate with chromosomes will be elucidated. The LANA domains required for episome persistence will be identified. The minimal cis-acting TR sequence requirements for LANA mediated episome persistence will be defined. Since episome persistence is critical for latent infection and most tumor cells are latently infected, this work may lead to new strategies for prevention and treatment of KSHV associated malignancies. Further, definition of the LANA and cis-acting element requirements for episome maintenance will enhance construction of LANA based gene therapy vectors.